Genetic analysis of feed quality and seed weight of sorghum inbred lines and hybrids using analytical methods and NIRS

Eight lines of grain sorghum and their F1 hybrids were evaluated for contents of crude protein (CP), fat (FAT), and starch (STA); protein digestibility (PD); and in vitro dry matter disappearance (IVDMD). The effect of seed weight (SW) on these traits and the potential use of near infrared reflectance spectroscopy (NIRS) to predict them also were investigated. The male lines included three normal-seeded lines (TX2737, TX435, and P954063) and two largeseeded lines (PL-1 and Eastin1). The female lines included commonU.S. seed parent lines (Wheatland, Redlan, and SA3042). The lines and their hybrids were grown under dryland conditions at Kansas State University experiment fields in Ashland and Belleville, Kansas, in 1999. The experiments were conducted using a randomized complete block design with four replications at each location. The effect of genotype was significant for all measured traits. The male parent lines were highly variable and expressed high levels of genetic variation in combining ability for CP, PD, STA, and SW. The female parents were genetically more uniform; however, significant general combining ability effects were noted for PD and SW. Significant negative correlations were noted between CP and STA and between SW and STA. Significant positive correlations were found between CP and SW and between FAT and IVDMD. Crude protein content was predicted accurately by NIRS. Fat content and IVDMD could not be predicted by NIRS. The NIRS equations based on ground samples were more accurate than those based on whole-seed samples

Genetic Analysis of Kafirins and Their Phenotypic Correlations with Feed Quality Traits, In Vitro Digestibility, and Seed Weight in Grain Sorghum

Twenty-three entries of grain sorghum (Sorghum bicolor (L.) Moench), including eight inbred lines (five males and three females) and 15 hybrids, were evaluated to determine the proportion of γ, αII, and β-αI-kafirins and their association with contents of crude protein, fat, and starch; protein digestibility; in vitro dry matter disappearance; and seed weight. The male lines included three normal-seeded lines (TX2737, TX435, and P954063) and two large-seeded lines (Eastin1 and PL-1). Female lines consisted of three common U.S. seed parent lines (Wheatland, Redlan, and SA3042). The lines and their hybrids were grown under dryland conditions at two locations in Kansas using a randomized complete block design. The effects of genotype, location, and males were significant for all kafirins. Wide variations in composition and general combining ability (GCA) for kafirin content were noted among parent lines and hybrids, with TX2737, Eastin1, and PL1 having the largest GCA values for γ (1.37), αII (1.99), and β-αI (2.57), respectively. Correlations among kafirins ranged from –0.89 to 0, whereas those of kafirins with feed quality traits, digestibility, and seed weight ranged from –0.45 to 0.48

Genetic Analysis of Kafirins and Their Phenotypic Correlations with Feed Quality Traits, In Vitro Digestibility, and Seed Weight in Grain Sorghum

Twenty-three entries of grain sorghum (Sorghum bicolor (L.) Moench), including eight inbred lines (five males and three females) and 15 hybrids, were evaluated to determine the proportion of γ, αII, and β-αI-kafirins and their association with contents of crude protein, fat, and starch; protein digestibility; in vitro dry matter disappearance; and seed weight. The male lines included three normal-seeded lines (TX2737, TX435, and P954063) and two large-seeded lines (Eastin1 and PL-1). Female lines consisted of three common U.S. seed parent lines (Wheatland, Redlan, and SA3042). The lines and their hybrids were grown under dryland conditions at two locations in Kansas using a randomized complete block design. The effects of genotype, location, and males were significant for all kafirins. Wide variations in composition and general combining ability (GCA) for kafirin content were noted among parent lines and hybrids, with TX2737, Eastin1, and PL1 having the largest GCA values for γ (1.37), αII (1.99), and β-αI (2.57), respectively. Correlations among kafirins ranged from –0.89 to 0, whereas those of kafirins with feed quality traits, digestibility, and seed weight ranged from –0.45 to 0.48

Population Improvement of Pearl Millet and Sorghum: Current Research, Impact and Issues for Implementation

Populations o f pearl millet and sorghum are being developed and improved fo r a variety o f purposes. In this paper, we present a global review o f current populations, their composition, and methods for improvement. The potential impact o f these programs is indicated by recent results regarding responses to recurrent selection and the linkages ofpopulation improvement with development o f lines and varieties in these two crops. Recent research on generating interpool populations and modeling responses to alternative recurrent selection methods are presented fo r population improvement o f pearl millet

Engineered Protein Nano-Compartments for Targeted Enzyme Localization

Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition

Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes

Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD(+). This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes

The life and scientific work of William R. Evitt (1923-2009)

Occasionally (and fortunately), circumstances and timing combine to allow an individual, almost singlehandedly, to generate a paradigm shift in his or her chosen field of inquiry. William R. (‘Bill’) Evitt (1923-2009) was such a person. During his career as a palaeontologist, Bill Evitt made lasting and profound contributions to the study of both dinoflagellates and trilobites. He had a distinguished, long and varied career, researching first trilobites and techniques in palaeontology before moving on to marine palynomorphs. Bill is undoubtedly best known for his work on dinoflagellates, especially their resting cysts. He worked at three major US universities and spent a highly significant period in the oil industry. Bill's early profound interest in the natural sciences was actively encouraged both by his parents and at school. His alma mater was Johns Hopkins University where, commencing in 1940, he studied chemistry and geology as an undergraduate. He quickly developed a strong vocation in the earth sciences, and became fascinated by the fossiliferous Lower Palaeozoic strata of the northwestern United States. Bill commenced a PhD project on silicified Middle Ordovician trilobites from Virginia in 1943. His doctoral research was interrupted by military service during World War II; Bill served as an aerial photograph interpreter in China in 1944 and 1945, and received the Bronze Star for his excellent work. Upon demobilisation from the US Army Air Force, he resumed work on his PhD and was given significant teaching duties at Johns Hopkins, which he thoroughly enjoyed. He accepted his first professional position, as an instructor in sedimentary geology, at the University of Rochester in late 1948. Here Bill supervised his first two graduate students, and shared a great cameraderie with a highly motivated student body which largely comprised World War II veterans. At Rochester, Bill continued his trilobite research, and was the editor of the Journal of Paleontology between 1953 and 1956. Seeking a new challenge, he joined the Carter Oil Company in Tulsa, Oklahoma, during 1956. This brought about an irrevocable realignment of his research interests from trilobites to marine palynology. He undertook basic research on aquatic palynomorphs in a very well-resourced laboratory under the direction of one of his most influential mentors, William S. ‘Bill’ Hoffmeister. Bill Evitt visited the influential European palynologists Georges Deflandre and Alfred Eisenack during late 1959 and, while in Tulsa, first developed several groundbreaking hypotheses. He soon realised that the distinctive morphology of certain fossil dinoflagellates, notably the archaeopyle, meant that they represent the resting cyst stage of the life cycle. The archaeopyle clearly allows the excystment of the cell contents, and comprises one or more plate areas. Bill also concluded that spine-bearing palynomorphs, then called hystrichospheres, could be divided into two groups. The largely Palaeozoic spine-bearing palynomorphs are of uncertain biological affinity, and these were termed acritarchs. Moreover, he determined that unequivocal dinoflagellate cysts are all Mesozoic or younger, and that the fossil record of dinoflagellates is highly selective. Bill was always an academic at heart and he joined Stanford University in 1962, where he remained until retiring in 1988. Bill enjoyed getting back into teaching after his six years in industry. During his 26-year tenure at Stanford, Bill continued to revolutionise our understanding of dinoflagellate cysts. He produced many highly influential papers and two major textbooks. The highlights include defining the acritarchs and comprehensively documenting the archaeopyle, together with highly detailed work on the morphology of Nannoceratopsis and Palaeoperidinium pyrophorum using the scanning electron microscope. Bill supervised 11 graduate students while at Stanford University. He organised the Penrose Conference on Modern and Fossil Dinoflagellates in 1978, which was so successful that similar meetings have been held about every four years since that inaugural symposium. Bill also taught many short courses on dinoflagellate cysts aimed at the professional community. Unlike many eminent geologists, Bill actually retired from actively working in the earth sciences. His full retirement was in 1988; after this he worked on only a small number of dinoflagellate cyst projects, including an extensive paper on the genus Palaeoperidinium